Targeted
next-generation
sequencing
in
the
diagnosis
of
neurodevelopmental disorders Nobuhiko Okamoto a, Fuyuki Miya b, Tatsuhiko Tsunoda b, Mitsuhiro Kato c, Shinji Saitoh d, Mami Yamasaki e, Atsushi Shimizu f, Chiharu Torii g, Yonehiro Kanemura h,i, Kenjiro Kosaki g a
Department of Medical Genetics, Osaka Medical Center and Research Institute for
Maternal and Child Health, Osaka, Japan b Laboratory for Medical Science Mathematics, Center for Integrative Medical Sciences, RIKEN, Yokohama, Japan. c Department of Pediatrics, Yamagata University Faculty of Medicine, Yamagata, Japan d Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan e Department of Pediatric Neurosurgery, Takatsuki General Hospital, Osaka, Japan f
Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank
Organization, Iwate Medical University, Iwate, Japan g Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cge.12492 This article is protected by copyright. All rights reserved
h
Division of Regenerative Medicine, Institute for Clinical Research, Osaka National
Hospital, National Hospital Organization, Osaka , Japan i Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan
Corresponding author
Nobuhiko Okamoto
This article is protected by copyright. All rights reserved
Department of Medical Genetics, Osaka Medical Center and Research Institute for Maternal and Child Health 840, Murodo-cho, Izumi, Osaka 594-1101, Japan Telephone number : +81-725-56-1220 Fax number : +81-725-56-5682 E-mail：
[email protected]
Conflict of interest The authors report no conflicts of interest.
Acknowledgments This study was supported by a grant from the Research on Applying Health Technology from the Ministry of Health, Labor and Welfare of Japan. We thank the patients and their families for participating in this work. We thank Mr. K. A. Boroevich for English proofreading. Abstract We developed a next-generation sequencing（NGS）based mutation screening strategy for neurodevelopmental diseases. Using this system, we screened 284 genes in 40 patients. Several novel mutations were discovered. Patient 1 had a novel mutation in ACTB. Her This article is protected by copyright. All rights reserved
dysmorphic feature was mild for Baraitser-Winter syndrome. Patient 2 had a truncating mutation of DYRK1A. She lacked microcephaly, which was previously assumed to be a constant feature of DYRK1A loss of function. Patient 3 had a novel mutation in GABRD gene. She showed Rett syndrome like features. Patient 4 was diagnosed with Noonan syndrome with PTPN11 mutation. He showed complete agenesis of corpus callosum. We discussed these novel findings.
Introduction
Despite many recent studies focusing on discovering the genetic basis of neurodevelopmental diseases, it is still largely unknown. We developed a next-generation sequencing（NGS）based mutation screening strategy. We screened 284 genes known or predicted to be associated with neurodevelopmental disorders with microcephaly/macrocephaly, CNS anomalies and ID.
Materials and Methods
We studied forty patients with neurodevelopmental disorders. They were negative for conventional cytogenetic studies and microarray analysis. With the approval of our
This article is protected by copyright. All rights reserved
institutional ethics committee, the patients were analyzed using this targeted sequencing. The genomic DNA of each patient was extracted from peripheral blood using extraction kit. Detail of the cell sample preparation was described in Supplemental methods.
Target gene sequencing Three μg of each sample DNA was sheared to 150-200bp using the Covaris DNA Shearing System (Wobum, MA, USA). To capture the target exonic DNA, we used the SureSelectXT Custom capture library (Agilent, Santa Clara, CA, USA) for 1.6 Mb of exons of neuronal gene capture. The sequence library was constructed with the SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing Library kit (Agilent) according to the manufacturer’s instructions. We performed DNA sequencing of either 76- or 101-bp paired-end reads using the Illumina Genome Analyzer IIx and HiSeq 2000 sequencer.
SNV calling NGS reads were aligned to the Human reference genome (GRCh37/hg19). We then excluded PCR duplicates, and extracted reads uniquely mapped to the reference genome that were properly paired within the insert size within mean + 2 SD of the mean. Base calling was performed in on-target regions, those regions within 100bp upstream and downstream of the exon capture probes. SNV and insertion and deletion (indel) calling were performed using
This article is protected by copyright. All rights reserved
SAM Tools and GATK software. We excluded known variants found in database. We then narrowed the candidates to only nonsynonymous, nonsense and splice site SNVs and frame shift indels. More details of method for variant calling were described in Supplementary methods.
NGS base-call quality check To analyze the quality of our base-calling algorithm, we used genotypes from HapMap database
(release
#28,
obtained
ftp://ftp.ncbi.nlm.nih.gov/hapmap/genotypes/2010-08_phaseII+III/).
Sanger
from sequence
validation of SNVs was performed using Applied Biosystems 3730xl DNA Analyzer (CA, USA).
Results To identify the causal mutation for neuronal diseases, we designed custom capture probes for the exons of 284 neuronal genes (Supplementary Table S1). We performed targeted genes sequencing using these probes and generated 1.7 Gb of sequence on average. The average read depth of the on-target regions was 608. To check the quality of our NGS base calls, we sequenced HapMap-JPT NA18943 using the same method as the other samples, and compared our NGS calls with the released genotype of the HapMap consortium. The
This article is protected by copyright. All rights reserved
genotypes for 3,129 locations were comparable between the two data sets. All but 16 of the 3,129 genotypes were concordant between our NGS calls and the HapMap data. We validated these mismatched 16 positions using Sanger sequencing and all 16 were consistent with our NGS calls (Supplementary Table S2 and S3). Based on this, we estimate the false positive and false negative rate of our SNV calling to be
next-generation
sequencing
in
the
diagnosis
of
neurodevelopmental disorders Nobuhiko Okamoto a, Fuyuki Miya b, Tatsuhiko Tsunoda b, Mitsuhiro Kato c, Shinji Saitoh d, Mami Yamasaki e, Atsushi Shimizu f, Chiharu Torii g, Yonehiro Kanemura h,i, Kenjiro Kosaki g a
Department of Medical Genetics, Osaka Medical Center and Research Institute for
Maternal and Child Health, Osaka, Japan b Laboratory for Medical Science Mathematics, Center for Integrative Medical Sciences, RIKEN, Yokohama, Japan. c Department of Pediatrics, Yamagata University Faculty of Medicine, Yamagata, Japan d Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan e Department of Pediatric Neurosurgery, Takatsuki General Hospital, Osaka, Japan f
Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank
Organization, Iwate Medical University, Iwate, Japan g Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cge.12492 This article is protected by copyright. All rights reserved
h
Division of Regenerative Medicine, Institute for Clinical Research, Osaka National
Hospital, National Hospital Organization, Osaka , Japan i Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan
Corresponding author
Nobuhiko Okamoto
This article is protected by copyright. All rights reserved
Department of Medical Genetics, Osaka Medical Center and Research Institute for Maternal and Child Health 840, Murodo-cho, Izumi, Osaka 594-1101, Japan Telephone number : +81-725-56-1220 Fax number : +81-725-56-5682 E-mail：
[email protected]
Conflict of interest The authors report no conflicts of interest.
Acknowledgments This study was supported by a grant from the Research on Applying Health Technology from the Ministry of Health, Labor and Welfare of Japan. We thank the patients and their families for participating in this work. We thank Mr. K. A. Boroevich for English proofreading. Abstract We developed a next-generation sequencing（NGS）based mutation screening strategy for neurodevelopmental diseases. Using this system, we screened 284 genes in 40 patients. Several novel mutations were discovered. Patient 1 had a novel mutation in ACTB. Her This article is protected by copyright. All rights reserved
dysmorphic feature was mild for Baraitser-Winter syndrome. Patient 2 had a truncating mutation of DYRK1A. She lacked microcephaly, which was previously assumed to be a constant feature of DYRK1A loss of function. Patient 3 had a novel mutation in GABRD gene. She showed Rett syndrome like features. Patient 4 was diagnosed with Noonan syndrome with PTPN11 mutation. He showed complete agenesis of corpus callosum. We discussed these novel findings.
Introduction
Despite many recent studies focusing on discovering the genetic basis of neurodevelopmental diseases, it is still largely unknown. We developed a next-generation sequencing（NGS）based mutation screening strategy. We screened 284 genes known or predicted to be associated with neurodevelopmental disorders with microcephaly/macrocephaly, CNS anomalies and ID.
Materials and Methods
We studied forty patients with neurodevelopmental disorders. They were negative for conventional cytogenetic studies and microarray analysis. With the approval of our
This article is protected by copyright. All rights reserved
institutional ethics committee, the patients were analyzed using this targeted sequencing. The genomic DNA of each patient was extracted from peripheral blood using extraction kit. Detail of the cell sample preparation was described in Supplemental methods.
Target gene sequencing Three μg of each sample DNA was sheared to 150-200bp using the Covaris DNA Shearing System (Wobum, MA, USA). To capture the target exonic DNA, we used the SureSelectXT Custom capture library (Agilent, Santa Clara, CA, USA) for 1.6 Mb of exons of neuronal gene capture. The sequence library was constructed with the SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing Library kit (Agilent) according to the manufacturer’s instructions. We performed DNA sequencing of either 76- or 101-bp paired-end reads using the Illumina Genome Analyzer IIx and HiSeq 2000 sequencer.
SNV calling NGS reads were aligned to the Human reference genome (GRCh37/hg19). We then excluded PCR duplicates, and extracted reads uniquely mapped to the reference genome that were properly paired within the insert size within mean + 2 SD of the mean. Base calling was performed in on-target regions, those regions within 100bp upstream and downstream of the exon capture probes. SNV and insertion and deletion (indel) calling were performed using
This article is protected by copyright. All rights reserved
SAM Tools and GATK software. We excluded known variants found in database. We then narrowed the candidates to only nonsynonymous, nonsense and splice site SNVs and frame shift indels. More details of method for variant calling were described in Supplementary methods.
NGS base-call quality check To analyze the quality of our base-calling algorithm, we used genotypes from HapMap database
(release
#28,
obtained
ftp://ftp.ncbi.nlm.nih.gov/hapmap/genotypes/2010-08_phaseII+III/).
Sanger
from sequence
validation of SNVs was performed using Applied Biosystems 3730xl DNA Analyzer (CA, USA).
Results To identify the causal mutation for neuronal diseases, we designed custom capture probes for the exons of 284 neuronal genes (Supplementary Table S1). We performed targeted genes sequencing using these probes and generated 1.7 Gb of sequence on average. The average read depth of the on-target regions was 608. To check the quality of our NGS base calls, we sequenced HapMap-JPT NA18943 using the same method as the other samples, and compared our NGS calls with the released genotype of the HapMap consortium. The
This article is protected by copyright. All rights reserved
genotypes for 3,129 locations were comparable between the two data sets. All but 16 of the 3,129 genotypes were concordant between our NGS calls and the HapMap data. We validated these mismatched 16 positions using Sanger sequencing and all 16 were consistent with our NGS calls (Supplementary Table S2 and S3). Based on this, we estimate the false positive and false negative rate of our SNV calling to be
